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receptors in cultured normal human epider- Paus R, Hoffmann U, Eichmu¨ller S, Czarnetzki BM nflammatory mediators in human keratino- mal . Biochem Biophys Res (1994) Distribution and changing density of cytes. J Pharmacol Exp Ther 304:217–22 Commun 291:124–9 gamma-delta T cells in murine skin during Stenn KS, Paus R (2001) Controls of hair follicle Ito T, Ito N, Bettermann A, Tokura Y, Takigawa M, the induced hair cycle. Br J Dermatol 130: cycling. Phys Rev 81:449–94 Paus R (2004) Collapse and restoration of 281–9 Sta¨nder S, Moormann C, Schumacher M, Budden- MHC class-I dependent immune privilege: Paus R, van der Veen C, Eichmu¨ller S, Kopp T, kotte J, Artuc M, Shpacovitch V et al. (2004) exploiting the human hair follicle as a model. Hagen E, Mu¨ller-Ro¨ver S et al. (1998) Expression of vanilloid receptor subtype 1 in Am J Pathol 164:623–34 Generation and cyclic remodeling of the cutaneous sensory fibers, mast cells, and hair follicle immune system in mice. J Invest Maurer M, Fischer E, Handjinski B, Von Stebut E, epithelial cells of appendage structures. Dermatol 111:7–18 Algermissen B, Bavandi A et al. (1997) Exp Dermatol 13:129–39 Activated skin mast cells are involved in Paus R, Mu¨ller-Ro¨ver S, Van der Veen C, Maurer Szallasi A, Blumberg PM (1999) Vanilloid (Cap- murine hair follicle regression (catagen). Lab M, Eichmu¨ller S, Ling G et al. (1999) A Invest 77:319–32 comprehensive guide for the recognition saicin) receptors and mechanisms. Pharma- col Rev 51:159–212 Mu¨ller-Ro¨ver S, Handjiski B, Van der Veen C, and classification of distinct stages of hair Eichmu¨ller S, Foitzik K, McKay IA et al. follicle morphogenesis. J Invest Dermatol Tominaga M, Caterina MJ, Malmberg AB, Rosen (2001) A comprehensive guide for the accu- 113:523–32 TA, Gilbert H, Skinner K et al. (1998) The rate classification of murine hair follicles in Peters EMJ, Handjiski B, Kuhlmei A, Hagen E, cloned capsaicin receptor integrates multiple distinct hair cycle stages. J Invest Dermatol Bielas H, Braun A et al. (2004) Neurogenic pain-producing stimuli. Neuron 21:531–43 117:3–15 inflammation in stress-induced termination Veronesi B, Oortgiesen M, Carter JD, Devlin RB Nakamura M, Sundberg JP, Paus R (2001) Mutant of murine hair growth is promoted by nerve (1999) Particulate matter initiates inflamma- laboratory mice with abnormalities in hair growth factor. Am J Pathol 165:259–71 tory cytokine release by activation of capsai- follicle morphogenesis, cycling, and/or struc- Southall MD, Li T, Gharibova LS, Pei Y, Nicol GD, cin and acid receptors in a human bronchial ture: annotated tables. Exp Dermatol 10: Travers JB (2003) Activation of epidermal epithelial cell line. Toxicol Appl Pharmacol 369–90 vanilloid receptor-1 induces release of proi- 154:106–15

Novel 14 Mutations in Patients with Severe Recessive Epidermolysis Bullosa Simplex

Journal of Investigative Dermatology (2006) 126, 1912–1914. doi:10.1038/sj.jid.5700312; published online 13 April 2006

TO THE EDITOR nant missense mutations; however, pa- many). Long-range polymerase chain Epidermolysis bullosa simplex (EBS) is tients with recessive EBS due to keratin reaction amplification of the KRT14 the most common subtype, accounting mutations have been reported, repre- was performed as described for one-half of all epidermolysis bullosa senting about 5% of all EBS mutations (Wood et al., 2003) and direct sequen- cases (Pfendner et al., 2005). It is (Porter and Lane, 2003). cing in both directions was performed, clinically characterized by nonscarring In this study, we investigated two using primers as published by Schui- blisters of the skin caused by little or no unrelated patients with severe neonatal lenga-Hut et al. (2003) and an ABI trauma, and morphologically by intra- blistering, both offspring of consangui- prism 3100 automated sequencer (ABI, epidermal blistering. The major subtypes neous, unaffected parents of Turkish Darmstadt, Germany). The study was of EBS result from mutations in either (patient 1), respectively German (pa- conducted according to the Declaration the (KRT5) or keratin 14 tient 2) origin. Indirect immunofluores- of Helsinki Principles, and the partici- (KRT14) gene, whereas mutations in cence (IIF) of the skin cryosections was pants gave their written informed con- the gene for (PLEC1) cause the performed as described (Hammami- sent. The medical committee of the rare forms, EBS with muscular dystro- Hauasli et al., 1998) with monoclonal University of Freiburg approved all phy and EBS Ogna. The clinical spec- antibodies anti-human keratin 5 (clone described studies. trum of EBS ranges from mild blistering D5/16 B4, Dako, Hamburg, Germany) Patient 1, a 2-year-old boy, showed of the hands and feet (EBS Weber–Cock- and keratin 14 (clone LL002, BioGenex, blistering predominantly on hands and ayne) to more generalized blistering San Ramon, CA). Genomic DNA was feet since birth (Figure 1a). In the course (EBS Koebner, EBS Dowling-Meara, and extracted from peripheral blood sam- of the disease, bullae became rarer, EBS with mottled pigmentation). EBS, ples collected from patients and their occurred mechanically induced also similarly to most of the keratin disorders unaffected parents using the Qiagen on the head and trunk and healed with- identified in humans, is caused by domi- Blood DNA Kit (Qiagen, Hilden, Ger- out scarring. Patient 2, aged 1 year, showed at birth extensive blistering of the hands and feet (Figure 1b) and suf- Abbreviations: EBS, epidermolysis bullosa simplex; IIF, indirect Immunofluorescence; KRT, keratin fered from congenital pneumonia. Oral

1912 Journal of Investigative Dermatology (2006), Volume 126 C Has et al. Novel Mutations in Recessive Epidermolysis Bullosa Simplex

a b and the short time they were available for observation. Absence of keratin 14 from the epidermis was caused by homozygous deletion/duplication mu- tations leading to premature termina- tion codons. Important information was provided by IIF staining of a skin biopsy: first, intraepidermal blistering c d was evident and second, the negative signal with keratin 14 antibodies per- mitted the rapid identification of the candidate gene. Except for the two novel mutations described here, 11 different KRT14 mutations associated with recessive EBS have been published before (Table 1): four with nonsense e f mutations, two with missense muta- tions, one with a splice site mutation, one compound heterozygous for non- sense and missense mutations, one with a deletion/insertion mutation, and two with deletion mutations. The less severely affected case reported by Batta et al. (2000) is the recessive ghc.427delC c.129dupC case with the shortest expressible KRT14 sequence to date, predicted to be truncated after only 30 amino acids (Porter and Lane, 2003; patient 6 in Table 1). In our patient 1, the truncation of keratin 14 is predicted to start after 143 amino acids, within the very beginning of the rod domain, and in Figure 1. Phenotypes of the patients, indirect immunofluorescence staining of skin sections and patient 2 after 44 amino acids, within mutations in the KRT14 gene. (a) Blisters and erosions on the feet of patient 1 at the age of 2 years. the head domain of keratin 14. Other (b) Patient 2 at 2 weeks of age showed severe blistering. (c) IIF with antibodies against keratin 5 showed intraepidermal splitting in the skin of patient 2. (d) IIF of control skin with antibodies against keratin 5. authors described severely affected (e) IIF staining with antibodies against keratin 14 yielded a negative signal in patient’s 1 skin. (f) IIF with keratin 14 ‘‘knockout’’ patients (Ta- antibodies against keratin 14 produced a positive signal in the basal layer in control skin. ble 1). However, no correlation be- Bar ¼ 100 mm (given in (f) but refers to all images). (g) Partial sequence of KRT14 exon 1, showing in the tween the position of the premature upper panel the mutation c.427delC (red arrow) and in the lower panel the wild-type sequence. (h) Partial termination codon and the severity of sequence of KRT14 exon 1, showing in the upper panel the mutation c.129dupC (red arrow) and in the the phenotype is obvious. Rather, these lower panel the wild-type sequence. mutations are more likely to be asso- ciated with nonsense-mediated mes- senger RNA decay. Chan et al. (1994) blisters were present in the first days. termination codon, two codons down- suggested that complete absence of a Later, he developed blisters on arms, stream, p.L143fsX2. In patient 2, also in keratin is less detrimental than the legs, and trunk after mechanical trau- exon 1 of KRT14, a homozygous disrupted filament assembly and aggre- ma. In both cases, IIF staining of skin duplication, c.129dupC was disclosed gate formation caused by dominant- sections revealed a split level through (Figure 1h), leading to a frame shift negative missense mutations. The pre- the basal cell layer (Figure 1c) and starting with codon 44 and premature sent findings and other reports in the antibodies against keratin 14 yielded termination codon 38 codons down- literature agree with this hypo- a negative staining (Figure 1e), suggest- stream (p.S44fsX38). The parents were thesis in that homozygous missense ing keratin 14 defects in both cases. found to be heterozygous carriers of the mutations lead to milder phenotypes Mutation detection in KRT14 revealed respective mutations. with blistering only in the extremities in patient 1 a homozygous one base In both patients, the clinical picture (patients 1, 10, and 11 in Table 1), pair deletion in exon 1, designa- was compatible with the diagnosis of whereas nonsense mutations are asso- ted c.427delC (Figure 1g); this results EBS Koebner. Mucosal involvement ciated with more generalized blistering in a frame shift starting with codon and other symptoms are difficult to (patients 2, 3, 4, 5, 7, 8, and 9 in 143 and formation of a premature assess because of the very young age Table 1).

www.jidonline.org 1913 C Has et al. Novel Mutations in Recessive Epidermolysis Bullosa Simplex

Table 1. KRT14 mutations in recessive EBS No. Phenotype, age Mutations References

1. Blistering affecting the lateral and dorsal aspects of the p.E144A/p.E144A Hovnanian et al. (1993) feet and plants, two patients aged 20 and 12 y 2. Severe generalized blistering, 5 y c.313_314delGC/ Rugg et al. (1994) c.313_314delGC 3. Generalized blistering, 29 m p.Y204X/p.Y204X Chan et al. (1994) 4. Severe generalized blistering, mucous membranes c.1842-2A4C/c.1842-2A4C Jonkman et al. (1996) occasionally affected; family with 4 patients aged 74, 67, 47, 34 y 5. Generalized blistering, occasional oral blisters, siblings of 7 and 6 y p.W305X/p.W305X Corden et al. (1998) 6. Mild generalized blistering, 18 m c.92delT/c.92delT Batta et al. (2000) p.I31fs86X 7. Generalized skin blistering and mild nail involvement c.744delC/insAG/ c.744delC/ Landschuetzer et al. (2003) and involvement of mucous membranes, 8 y insAG p.Y248X 8. Widespread blistering and oral mucosal involvement, p.Q396X/p.Q396X Ciubotaru et al. (2003) anaemia and failure to thrive in the first year of life, NA 9. Widespread blistering over palms and soles and oral p.W305X/p.W305X Ciubotaru et al. (2003) and genital mucosa, NA 10. Blisters over palms and soles, NA p.Q396X/p.R388H Ciubotaru et al. (2003) 11. Blisters over hands and feet, 4 y p.R134C/p.R134C Indelman et al. (2005) 12. Blisters on hands and feet, later generalized, 2 y c.427delC/c.427delC This study p.L143fsX2 13. Severe blistering of hands and feet, oral erosions, c.129dupC/c.129dupC This study widespread induced blisters, 1 y p.S44fsX38 m, months; NA, not available; y, years.

CONFLICT OF INTEREST and a function for an sive epidermolysis bullosa simplex. J Invest The authors state no conflict of interest. . Dev 8:2574–2587 Dermatol 107:764–9 Ciubotaru D, Bergman R, Baty D, Indelman M, Landschuetzer CM, Klausegger A, Pohla-Gubo G, ACKNOWLEDGMENTS Pfendner E, Petronius D et al. (2003) Epidermo- Hametner R, Richard G, Uitto J et al. (2003) We thank the patients and their families and the lysis bullosa simplex in Israel. Arch Dermatol A novel homozygous nonsense deletion/ partners of the German Netzwerk Epidermolysis 139:498–505 insertion mutation in the keratin 14 gene bullosa (www.netzwerk-eb.de) for referring pa- (Y248X; 744delC/insAG) causes recessive Corden L, Mellerio J, Gratian MJ, Eady AJ, Harper tients for mutation analysis. This work was epidermolysis bullosa simplex Ko¨bner. Clin JI, Lacour M et al. (1998) Homozygous supported in part by the EB-Network grant from Exp Dermatol 28:77–9 nonsense mutation in helix 2 of K14 causes the German Ministry for Research and Education Pfendner EG, Sadowski SG, Uitto J (2005) Epider- severe recessive epidermolysis bullosa sim- (BMBF). molysis bullosa simplex: recurrent and de plex. Hum Mutat 11:279–85 1 1 novo mutations in the KRT5 and KRT14 genes, Cristina Has , Yow-Ren Chang , Hammami-Hauasli N, Schumann H, Raghunath phenotype/genotype correlations and impli- Andreas Volz1, Doris Hoeping1, M, Kilgus O, Luthi U, Luger T et al. (1998) cations for genetic counselling and prenatal Ju¨rgen Kohlhase2 and Leena Some, but not all, glycine substitution muta- diagnosis. JInvestDermatol125:239–43 Bruckner-Tuderman1 tions in COL7A1 result in intracellular Porter R, Lane B (2003) Phenotypes, genotypes accumulation of collagen VII, loss of anchor- and their contribution to understanding 1 Department of Dermatology, University of ing fibrils, and skin blistering. J Biol Chem keratin function. Trends Genet 19:278–85 Freiburg, Freiburg, Germany and 2Institute 273:19228–34 Rugg EL, McLean WH, Lane EB, Pitera R, of Human Genetics and Anthropology, Hovnanian A, Pollack E, Hilal L, Rochat A, Prost McMillan JR, Dopping-Hepenstal PJC et al. University of Freiburg, Freiburg, Germany. C, Barrandon Y et al. (1993) A missense (1994) A functional ‘‘knockout’’ of human E-mail: [email protected] mutation in the rod domain pf keratin 14 asso- keratin 14. Genes Dev 8:2563–73 ciated with recessive epidermolysis bullosa Schuilenga-Hut PH, Vlies P, Jonkman MF, REFERENCES simplex. Nat Genet 3:327–32 Waanders E, Buys CH, Scheffer H (2003) Mutation analysis of the entire keratin 5 and Batta K, Rugg EL, Wilson NJ, West N, Goodyear Indelman M, Bergman R, Sprecher E (2005) A 14 genes in patients with epidermolysis novel recessive missense mutation in KRT14 H, Lane EB et al. (2000) A keratin 14 bullosa simplex and identification of novel reveals striking phenotypic heterogeneity ‘knockout’ mutation in recessive epidermo- mutations. Hum Mutat 21:447 lysis bullosa simplex resulting in less severe in epidermolysis bullosa simplex. J Invest Wood P, Baty DU, Lane EB, McLean WH (2003) disease. Br J Dermatol 143:621–7 Dermatol 124:272–4 Long-range polymerase chain reaction for Chan Y, Anton-Lamprecht I, Yu QC, Jackel A, Jonkman MF, Heeres K, Pas HH, Van Luyn MJA, specific full-length amplification of the Zabel B, Ernst JP et al. (1994) A human Elema JD, Corden LD et al. (1996) Effects of human keratin 14 gene and novel keratin keratin 14 ‘‘knockout’’: the absence of K14 keratin 14 ablation on the clinical and 14 mutations in epidermolysis bullosa sim- leads to severe epidermolysis bullosa simplex cellular phenotype in a kindred with reces- plex patients. J Invest Dermatol 120:495–7

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